Hydrophobic ion pair for the oral delivery of leucine-enkephalin

1.Purpose The global therapeutic peptides market was valued at US$26.98 billion in 2019 and is projected to reach US$51.24 billion by 2027, growing at a compound annual growth rate (CAGR) of 8.7% from 2020 to 2027 (Research 2021). The peptide market is bound to grow by the increase in metabolic diseases andcancers but their translation remains challenging due to pre-systemic degradation, short plasma half-lives, and poor permeability across physiological barriers. Ion-pairing has been proposed as a method for the oraldelivery of peptides (Griesser, Hetenyi et al. 2017) as it enables increase stability to gastrointestinal enzymedegradation and enhanced permeability across physiological barriers. In this study, we explore the formationof an ion-pair of leucine-enkephalin (LENK), an endogenous opioid pentapeptide with applications as an oraltherapeutic for the treatment of pain (Lalatsa, Lee et al. 2012), Chron’s disease, and other gastrointestinalinflammatory conditions, as a strategy to enhance its oral bioavailability (Owczarek, Cibor et al. 2011). 2.Method Standard orthogenic solid phase peptide synthesis was utilized to synthesize LENK (0.5 mmole scale) (Lalatsa, Lee et al. 2012) and the peptide was obtained in high yield ( >85%) and highpurity ( >95%) as determined by HPLC and LC-MS. LENK (2.73 mmole, 1.52 mg/mL, 3mL) andsodium docusate (3mL) in deionized water (pH: 2.9) were mixed at different molar ratio (1:1,1:3, 1:5 respectively) to understand optimal ratio for pair formation and vortexed over 1 minute prior centrifugation or ultracentrifugation for 90 minutes at 4oC at 40,000 rpm (Hitachi Ultracentrifuge CP1000 NX). The supernatant was separated and the pellet was frozen withliquid nitrogen and lyophilized for 24 hours (Teslar, -50oC, 0.2mbar pressure). The LENK content was characterized by a previously validated HPLC method (Lalatsa, Lee et al. 2012). Intestinal fluid (50mM phosphate buffer, pH 6.6) was prepared from excised mouse intestine(C57BL/6, 8 weeks old, male) as previously described and characterized for protein contentusing the Bradford assay and diluted (1mg mL) (Lalatsa, Lee et al. 2012). Stability studies (37oC, 50 rpm) were undertaken and the remaining LENK was analyzed using HPLC after dilution in ice-cold acetonitrile (1:1) and centrifugation. Permeability studies across a Caco-2 cell monolayer were undertaken for LENK, the ion-paired LENK, and antipyrine with FITC dextran (3-5 kDa) as an internal control as previously described (Hubatsch, Ragnarsson et al. 2007). Caco-2 seed density was 10,000 cells/cm in this experiment. 3.Results The 1:1 and the 1:5 ratios resulted in low ion-pairing yields of 37% and 19%. The optimal ratio for pairing was identified to be 1:3 which resulted in an ion-pairing yield of 56% and this ratio was further tested in stability and permeability studies. Intestinal wash was selected as it more closely describes in vivo data scenarios (McConnell, Basit et al. 2008). LENK degraded rapidly in the intestinal wash (< 60 minutes) while the ion pair showed a 3.5-fold increase in half-life and showed levels that were significantly different after the first initial 10 minutes (Student t-test, p< 0.05) (Figure 1). Permeability across the Caco-2 cells indicate a trend for higher uptake for ion-pair LENK, but due to low TEER values obtained in our experiments due to low cell seed density (Figure 2). 4.Conclusion Ion-paired LENK has shown a significant enhancement in oral gastrointestinal stability and further studies are underway to assess its oral bioavailability across Caco-2 monolayers. Combining ion-pair technology with solid state or additively manufactured formulations can enable the production of an oral LENK formulation for the treatment of pain and inflammatory diseases such as Chron’s disease

Continuous microfluidic manufacture of cocrystals using 3D printed chips coupled with spray coating

Cocrystals have emerged as a promising strategy to improve the physicochemical properties of active pharmaceutical ingredients (APIs) by forming a new crystalline phase from two or more components. Particle size and morphology control are key quality attributes for cocrystal medicinal products. The needle-shaped morphology is often considered high risk and complex in the manufacture of solid dosage forms. Co-crystal particle engineering requires advanced methodologies to ensure high-purity cocrystals with improved soubility and bioavailability and with optimal crystal habit for industrial manufacturing. In this study, 3D-printed microfluidic chips were used to control the cocrystal habit and polymorphism of the sulfadimidine (SDM): 4-aminosalicylic acid (4ASA) cocrystal. The addition of PVP in the aqueous phase during mixing resulted in a high-purity cocrystal (with no traces of the individual components), while it also inhibited the growth of needle-shaped crystals. When mix-tures were prepared at the macroscale, PVP was not able to control the crystal habit and impurities of individual mixture components remained, indicating that the micro-fluidic device allowed for a more homogenous and rapid mixing process controlled by the flow rate and the high surface-to-volume ratios of the microchannels. Continuous manufacturing of SDM:4ASA cocrystals coated on beads was successfully implement-ed when the microfluidic chip was connected in line to a fluidized bed allowing co-crystal formulation generation by mixing

Understanding Direct Powder Extrusion for Fabrication of 3D Printed Personalised Medicines: A Case Study for Nifedipine Minitablets

Fuse deposition modelling (FDM) has emerged as a novel technology for manufacturing 3D printed medicines. However, it is a two-step process requiring the fabrication of filaments using a hot melt extruder with suitable properties prior to printing taking place, which can be a rate-limiting step in its application into clinical practice. Direct powder extrusion can overcome the difficulties encountered with fabrication of pharmaceutical-quality filaments for FDM, allowing the manufacturing, in a single step, of 3D printed solid dosage forms. In this study, we demonstrate the manufacturing of small-weight (<100 mg) solid dosage forms with high drug loading (25%) that can be easily undertaken by healthcare professionals to treat hypertension. 3D printed nifedipine minitablets containing 20 mg were manufactured by direct powder extrusion combining 15% polyethylene glycol 4000 Da, 40% hydroxypropyl cellulose, 19% hydroxy propyl methyl cellulose acetate succinate, and 1% magnesium stearate. The fabricated 3D printed minitablets of small overall weight did not disintegrate during dissolution and allowed for controlled drug release over 24 h, based on erosion. This release profile of the printed minitablets is more suitable for hypertensive patients than immediate-release tablets that can lead to a marked burst effect, triggering hypotension. The small size of the minitablet allows it to fit inside of a 0-size capsule and be combined with other minitablets, of other API, for the treatment of complex diseases requiring polypharmacy within a single dosage form

Development of Advanced 3D-Printed Solid Dosage Pediatric Formulations for HIV Treatment

The combination of lopinavir/ritonavir remains one of the first-line therapies for the initial antiretroviral regimen in pediatric HIV-infected children. However, the implementation of this recommendation has faced many challenges due to cold-chain requirements, high alcohol content, and unpalatability for ritonavir-boosted lopinavir syrup. In addition, the administration of crushed tablets has shown a detriment for the oral bioavailability of both drugs. Therefore, there is a clinical need to develop safer and better formulations adapted to children’s needs. This work has demonstrated, for the first time, the feasibility of using direct powder extrusion 3D printing to manufacture personalized pediatric HIV dosage forms based on 6 mm spherical tablets. H-bonding between drugs and excipients (hydroxypropyl methylcellulose and polyethylene glycol) resulted in the formation of amorphous solid dispersions with a zero-order sustained release profile, opposite to the commercially available formulation Kaletra, which exhibited marked drug precipitation at the intestinal pH

Engineering 3D printed microfluidic chips for the fabrication of nanomedicines

Currently, there is an unmet need to manufacture nanomedicines in a continuous and controlled manner. Three-dimensional (3D) printed microfluidic chips are an alternative to conventional PDMS chips as they can be easily designed and manufactured to allow for customized designs that are able to reproducibly manufacture nanomedicines at an affordable cost. The manufacturing of microfluidic chips using existing 3D printing technologies remains very challenging because of the intricate geometry of the channels. Here, we demonstrate the manufacture and characterization of nifedipine (NFD) polymeric nanoparticles based on Eudragit L-100 using 3D printed microfluidic chips with 1 mm diameter channels produced with two 3D printing techniques that are widely available, stereolithography (SLA) and fuse deposition modeling (FDM). Fabricated polymeric nanoparticles showed good encapsulation efficiencies and particle sizes in the range of 50–100 nm. SLA chips possessed better channel resolution and smoother channel surfaces, leading to smaller particle sizes similar to those obtained by conventional manufacturing methods based on solvent evaporation, while SLA manufactured nanoparticles showed a minimal burst effect in acid media compared to nanoparticles fabricated with FDM chips. Three-dimensional printed microfluidic chips are a novel and easily amenable cost-effective strategy to allow for customization of the design process for continuous manufacture of nanomedicines under controlled conditions, enabling easy scale-up and reducing nanomedicine development times, while maintaining high-quality standards

3D printing technologies in personalized medicine, nanomedicines, and biopharmaceuticals

3D printing technologies enable medicine customization adapted to patients' needs. There are several 3D printing techniques available, but majority of dosage forms and medical devices are printed using nozzle-based extrusion, laser-writing systems, and powder binder jetting. 3D printing has been demonstrated for a broad range of applications in development and targeting solid, semi-solid and locally applied or implanted medicines. 3D printed solid dosage forms allow the combination of one or more drugs within the same solid dosage form to improve patient compliance, facilitate deglutition, tailor the release profile, or fabricate new medicines for which no dosage form is available. Sustained release 3D-printed implants, stents and medical devices have been used mainly for joint replacement therapies, medical prostheses, and cardiovascular applications. Locally applied medicines such as wound dressing, microneedles, and medicated contact lenses have also been manufactured using 3D printing techniques. The challenge is to select the 3D printing tech-nique most suitable for each application and the type of pharmaceutical ink that should be devel-oped that possesses the required physicochemical and biological performance. The integration of biopharmaceuticals and nanotechnology-based drugs along with 3D printing ("Nanoprinting") brings printed personalized nanomedicines within the most innovative perspectives for the coming years. Continuous manufacturing through the use of 3D-printed microfluidic chips facilitates their translation into clinical practice

Artificial intelligence (AI) applications in drug discovery and drug delivery : revolutionizing personalized medicine

Artificial intelligence (AI) encompasses a broad spectrum of techniques that have been utilized by pharmaceutical companies for decades, including machine learning, deep learning, and other advanced computational methods. These innovations have unlocked unprecedented opportunities for the acceleration of drug discovery and delivery, the optimization of treatment regimens, and the improvement of patient outcomes. AI is swiftly transforming the pharmaceutical industry, revolutionizing everything from drug development and discovery to personalized medicine, including target identification and validation, selection of excipients, prediction of the synthetic route, supply chain optimization, monitoring during continuous manufacturing processes, or predictive maintenance, among others. While the integration of AI promises to enhance efficiency, reduce costs, and improve both medicines and patient health, it also raises important questions from a regulatory point of view. In this review article, we will present a comprehensive overview of AI’s applications in the pharmaceutical industry, covering areas such as drug discovery, target optimization, personalized medicine, drug safety, and more. By analyzing current research trends and case studies, we aim to shed light on AI’s transformative impact on the pharmaceutical industry and its broader implications for healthcare

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Recent smell loss is the best predictor of COVID-19 among individuals with recent respiratory symptoms

In a preregistered, cross-sectional study we investigated whether olfactory loss is a reliable predictor of COVID-19 using a crowdsourced questionnaire in 23 languages to assess symptoms in individuals self-reporting recent respiratory illness. We quantified changes in chemosensory abilities during the course of the respiratory illness using 0-100 visual analog scales (VAS) for participants reporting a positive (C19+; n=4148) or negative (C19-; n=546) COVID-19 laboratory test outcome. Logistic regression models identified univariate and multivariate predictors of COVID-19 status and post-COVID-19 olfactory recovery. Both C19+ and C19- groups exhibited smell loss, but it was significantly larger in C19+ participants (mean±SD, C19+: -82.5±27.2 points; C19-: -59.8±37.7). Smell loss during illness was the best predictor of COVID-19 in both univariate and multivariate models (ROC AUC=0.72). Additional variables provide negligible model improvement. VAS ratings of smell loss were more predictive than binary chemosensory yes/no-questions or other cardinal symptoms (e.g., fever). Olfactory recovery within 40 days of respiratory symptom onset was reported for ~50% of participants and was best predicted by time since respiratory symptom onset. We find that quantified smell loss is the best predictor of COVID-19 amongst those with symptoms of respiratory illness. To aid clinicians and contact tracers in identifying individuals with a high likelihood of having COVID-19, we propose a novel 0-10 scale to screen for recent olfactory loss, the ODoR-19. We find that numeric ratings ≤2 indicate high odds of symptomatic COVID-19 (4&lt;10). Once independently validated, this tool could be deployed when viral lab tests are impractical or unavailable

Engineering 3D printed microfluidic chips for the fabrication of nanomedicines

1.Purpose -- Nanomedicine manufacture remains expensive and difficult to scale-up which limits the uptake of nano-enabled technologies by industry. Thus, there is an urgent unmet need for continuous and controlled manufacturing processes. Microfluidic manufacture has emerged as a novel and easily adaptable strategy to overcome these challenges, but majority of chips used are fabricated using polydimethulsiloxane (PDMS) and soft mask lithography that remains tedious, not easily customizable and requires specialized equipment and expertise for their production. 3D printed chips are a novel and easily adaptable cost-effective alternative able to provide microfluidic chips for enabling quick pilot studies towards the manufacture of nanomedicines under controlled conditions with optimal and controlled characteristics enabling easy scale-up and shorter development times. However, for 3D printed chips to be successful as an alternative, 3D printing channels with adequate resolution to produce the required geometry needs to be demonstrated. In this work, we utilized the most easily accessible 3D printing techniques (fused deposition modelling (FDM) and sterolithography (SLA)) and commercially available solvent resistant filaments and resin to produce designed microfluidic chips with appropriate geometry and channel characteristics to allow for the manufacture of polymeric nanoparticles based on polymethacrylate polymers encapsulating high concentration of a BCS class II drug (nifedipine, NFD). 2.Method -- Chips were designed in Tinkercad® (Autodesk®) and measured 8.2 cm in length, 3.5 cm in width, and 0.7 cm in height. Channel length was 44 cm and the diameter was 1 mm. Chip designed was exported into a standard tessellation language (.stl) digital file. An Anycubic Mega Zero FDM printer printed 70 layers of the microfluidic chip at 245°C, with a 0.4 mm diameter nozzle, 0.1 mm layer height, and 10 mm/s printer and 30 mm/s travel speed with cyclic olefin copolymer filament. The Anycubic Photon Mono X (LCD-based SLA printer with 405 nm light source and 0.01 mm Z resolution) was used for stereolithography. Anycubic® UV sensitive resin (transparent yellow) was photopolymerized at 405 nm. The print settings were 0.05 mm layer height, 60 s bottom exposure, 3 s normal exposure, 1 s off-time, and 140 layers. Polymeric nifedipine loaded nanoparticles were prepared using solvent evaporation and microfluidically. For the latter, the aqueous phase (8 ml) consisting of Tween 80 in deionized water (0.25 % w/v) and the organic phase consisting of Eudragit L100-55 (30 mg) and NFD (10 mg) dissolved in ethanol (2 ml) were loaded into two 10 ml syringes. Using two syringe pumps (New Era Pump Systems, NY, USA), the organic phase was flown at a rate of 0.5 ml min-1 and the aqueous phase at 2 ml min-1. The eluate was rota-evaporated for 10 minutes at 150 rpm and 60 °C to remove the ethanol and centrifuged at 5,000 rpm for 5 minutes to remove any free NFD. Part of the supernatant was lyophilized for 24 hours under 0.2 mbar pressure at -50oC. Formulations were characterized in terms of drug loading, particle size, zeta potential and morphology and the channels were imaged with light microscopy, scanning electron microscopy while the surface roughness was measured with profilometry. Solid state characterisation of lyophilized particles were also undertaken. Release of NFD from nanoparticles was assessed using a type II dissolution apparatus (Ewerka DT 80, Heusenstamm, Germany) under simulated gastric and intestinal media (Ayyoubi S.). 3.Results --  The chip geometry produced was in close accordance to the .stl file sent for printing (Fig. 1a). The channel diameter ranged from 985 – 1015 µm. SLA-printed chips exhibited channels with a smoother surface (10.5-fold) than FDM chips. NFD nanoparticles showed a 7% greater drug encapsulation when manufactured by SLA than with FDM chips (one-way ANOVA, p < 0.05) which was closer to the loading reported by solvent evaporation. NFD nanoparticles manufactured using SLA chips were significantly smaller than those particles obtained from FDM chips, 68 ± 1 nm versus 75 ± 1 nm, respectively (one-way ANOVA, p < 0.05), which was closer to the particle size obtained by solvent evaporation (Fig. 2). Lyophilised nanoparticles showed similar FTIR, pXRD, and DSC patterns obtained from both SLA and FDM chips. NFD release was hampered in acidic media (<20% at 1 hour), but near complete released was achieved when the pH was raised to 6.8 within 6 hours, which was similar to that obtained for particles prepared with solvent evaluation (Fig. 3). However, NFD particles produced with FDM showed a burst release in acidic media (~40%) followed by controlled release in simulated intestinal media (p<0.05, One-way Anova). NFD localization within the particles produced with different 3D printed chips due differences in surface roughness and drug–polymer interactions are contributing to these findings. The smoother channels of SLA chips lead to a more homogenous loading process, where NFD is located within the core of the polymeric nanoparticles, which is further supported by the smaller particle size and controlled release profile in acidic media where NFD is more likely to be soluble.  4. Conclusion -- 3D printed microfluidic chips with 1 mm diameter channels have been successfully designed and manufactured and are capable to engineer polymeric nanoparticles with good encapsulation efficiencies and particle sizes of ~100 nm, like nanoparticles obtained by solvent evaporation. 3D printed microfluidic chips control the process and convert discontinuous methods into a continuous nanomedicine manufacturing process that are easily industrialized